Elucidating the An. funestus species complex Since 2000, renewed international commitment to malaria control has led to a substantial increase in malaria intervention coverage globally. However, there are disturbing signs that this trend has slowed, stalled or even reversed in some regions due to failures in vector control. Even if all households had insecticidal bed nets, emerging insecticide resistance, behavioral changes and outdoor transmission by vectors raise concerns that existing tools are inadequate to eliminate or even interrupt intense malaria transmission. Near-term strategies to manage resistance have been outlined, but longer-term research into new anti-vector interventions to complement existing tools is imperative. Many basic aspects of anopheline biology that could contribute to more effective control remain poorly defined. Anopheles funestus is one of the three most important and widespread vectors of human malaria in tropical Africa, but unlike An. gambiae, it is understudied. This major vector conceals a group of close relatives that are morphologically similar or identical as adults. None of these species, with one exception, has been directly implicated as a secondary vector. However, they can rest indoors in large numbers and are considered important due to their impact on scarce resources available for vector control. They overlap with An. funestus, but their geographic distribution is largely unknown, and their actual or potential contribution to malaria transmission is also uncertain. Traditionally characterized as zoophilic and exophilic, their behavior is plastic and depends upon host availability and other (unknown) environmental factors. Moreover, the taxonomic complexity of this group is at least as high as the better- known An. gambiae complex; within each named (or unnamed) species are genetically heterogeneous clusters whose interrelationships have never been resolved, and additional cryptic diversity is being reported by vector control programs. The central goal of this R21 is to elucidate the An. funestus complex. Toward this end, we propose two specific Aims: (1) Generate reference genome assemblies for species in the An. funestus complex. Complete reference genome assemblies have the power necessary to elucidate taxonomic and phylogenetic relationships among closely related taxa. Traditional approaches to genome assembly are impractical, but our preliminary data establish that the DISCOVAR de novo algorithm represents a simple, rapid and low-cost solution. (2) Assess sequence divergence and speciation history in the An. funestus complex. Using the new reference assemblies and additional light whole genome sequencing (~12 samples/taxon), we will analyze genome-wide SNP data to construct an initial portrait of population genomic patterns and species relationships for five members of the An. funestus complex. This project will provide the necessary frame of reference for species delimitation, identification, and vector incrimination in the An. funestus complex to support sustainable control or elimination of malaria transmission in Africa.